Galls on Alstonia scholaris leaves as air pollution indicator … · Galls on Alstonia scholaris leaves as air pollution indicator Partha Talukdar1, Kaushiki Das2, Shrinjana Dhar2,

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WSN 52 (2016) 181-194 EISSN 2392-2192

Galls on Alstonia scholaris leaves as air pollution indicator

Partha Talukdar1, Kaushiki Das2, Shrinjana Dhar2, Soumendra Nath Talapatra2

and Snehasikta Swarnakar3,* 1Department of Botany, Srirampore College, University of Calcutta,

William Carey Road, Hooghly, West Bengal, India

2Career Advancement Solutions, Maheshtala, Kolkata – 700142, India

3Cancer Biology and Inflammatory Disorders Division, CSIR - Indian Institute of Chemical Biology,

4 Raja S.C. Mullick Road, Kolkata – 700032, India

*E-mail mail address: [email protected] ; *Phone: +913324995759

ABSTRACT

Air pollution arises mainly from automobiles and industries is well known fact. Monitoring and

detection by instrument cannot be possible everywhere however, indication from plant species by their

alterations in leaf morphology and anatomy may be a suitable easy screening measurement. The

present study aims to detect morphological features with special reference to gall quantification and

anatomy of leaves of Alstonia scholaris R. Br., found in eastern Indian urban and suburban area that

are exposed to vehicular emission. The results indicated alterations of leaf morphology along with

length (L), breadth (B), L/B ratio and significantly increased (P < 0.001, 0.01 and 0.05) Air Pollution

Index (API). It is concluded that vehicular emission can be monitored as an early indication through

increased API in A. scholaris. Further research would be needed in relation to secondary metabolites

alteration, biochemical and genetic parameters to know pollutant susceptibility as an indicator. In

addition, anatomical abnormalities in gall formation as well as numbers were also pronounced in

leaves exposed to various load of air pollution.

Keywords: Automobile air pollution; gall formation; bioindicator plant; leaf morphology and anatomy;

Air Pollution Index (API); Alstonia scholaris

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1. INTRODUCTION

Air pollutants generate from different activities viz. automobiles, fugitive and stack,

burning of solid wastes etc. Beside these, automobiles cause both primary and secondary air

pollutants in India, which has already been reported by regulatory authorities (CPCB, 2009;

Citizen’s Report, 2011) and several researchers (Joshi and Swami, 2007; Diwvedi et al.,

2008).

It has been established that air pollution impact on the plant species worldwide in

relation to plant-environment interactions, since the plants are more sensitive as well as

resistant species compare to other organisms. The abnormalities are including plant external

morphology, anatomy, physiology and/or biochemical profiles indicate about the polluted

environment. The air pollutants response varies in plants as species to species and also in

terms of different types, reaction mechanisms, concentration and exposure time. The

pollutants when enter into the plants then react before being removed or absorbed and may

lead to accumulation, chemical transformation and incorporation into the metabolism system.

In these processes, some plants are injured while others show minimal effects or no impacts

(Choudhury and Banerjee, 2009).

Among other trees, Devil tree, Alstonia scholaris, R. Br. (Apocynaceae) is an

evergreen, tropical tree with white funnel-shaped flowers and milky sap and grows to height

of 30-40 m found in most of parts of India. Among medicinal properties, A. scholaris is also

used as an avenue tree because this plant is prescribed in greenbelt and easy to manage in

polluted areas, requires less water and bioindicator plant (Muhammad et al., 2014; Mandal

Biswas et. al. 2014). The plant species under greenbelt can effectively be prescribed as air

pollutants prevention as resistant (tolerant) and sensitive or responding (Warren, 1973; Singh

and Rao, 1983; Tiwari and Tiwari, 2006). The gall formation rate is higher when plant species

are susceptible to air pollutants and induce the nitrogen content in the leaves then improve the

herbivore palatability. Furlan et al. (2004) have hypothesized in Tibouchina pulchra (Cham.)

Cogn. (Melastomataceae), a native species of the Brazilian Atlantic forest has pollution-

resistant capacity. According to them, when stress caused by air pollutants, the levels of

secondary metabolites decreased, while the contents of nitrogen increased, due to a partial

blockage of primary metabolism participated in the of protein and other nitrogenous

compounds synthesis. They found the reduced amounts of secondary metabolites and the

increased amounts of foliar nitrogen, have found the potential to enhance herbivore foliar

damage.

Generally the leaf galls of A. scholaris induced by a bug, Pauropsylla tuberculata

crawf., which is an insect (class Psyllidae, order homoptera) as reported by researchers

(Hodkinson, 1984; Mandal Biswas et al., 2014). The research work has revealed that

formation of galls induced by homoptera insects, correlated with the feeding habit and

predominantly inhabited in leaf galls (Meyer, 1987). The insects are known to extract

nutrients from the phloem, xylem or non-conducting plant cell (Arya et. al., 1975). The insect

activates a perturbation in growth mechanisms and alters the cellular differentiation processes

of the leaf in the host plant, modifying the plant architecture as per its advantage (Raman,

2007).

Many studies as bioindicator plants showing visible leaf damages, morphology,

anatomy and biochemical changes related to air pollution internationally (Middleton et al.,

1956; Bull and Mansfield, 1974; Husen et al., 1999; Naveed et al., 2010; Seyyed and


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Koochak 2011) as well as nationally (Tiwary et al., 2008; Saquib et al., 2010; Deepalakshmi,

2013; Nandy et al., 2014). The previous studies have emphasized on alteration of physico-

chemical parameters by air pollution. On the other hand, different studies carried out on foliar

galls morphology, anatomy and biochemistry after insect (Pauropsylla tuberculata) infection

and deposition their eggs (Albert et al., 2011; Mandal Biswas et al., 2014).

The present study attempts to know the morphological and anatomical deformities along

with galls quantification on leaves of A. scholaris found near roadside in eastern India.

2. MATERIALS AND METHODS

2. 1. Study area

The study areas were selected as per vehicular loads. The study was carried out at 4

sampling stations viz. (i) low vehicular load (LVL) as control area, (ii) moderate vehicular

load (MVL), high vehicular load (HVL) and (iii) heavy vehicular load (HeVL) as

experimental area. These four sampling stations were selected on the basis of low, moderate,

high and heavy traffic density along with vehicular movement as per visualization. The plant

species was selected Alstonia scholaris R. Br. growing near roadside of above mentioned area

because gall formations are more common in this species.

The affected leaf shape was determined by the study of shape as length (L), breadth (B)

and L/B ratio and anatomy of leaves along with galls. It was also studied number of galls per

leaf. All the leaves randomly selected from 5 trees of above-mentioned area.

2. 2. Area of Leaves

The 10 leaves were collected randomly from per tree of above-mentioned area.

Individual leaf was cleaned properly in running water and soaked with blotting paper. The

area of leaves especially L/B (Length / Breadth) ratio of leaf (in cm), was measured manually.

2. 3. Galls quantification

The 10 leaves per tree were collected randomly. Individual leaf was cleaned properly in

running water and soaked with blotting paper. The quantification of galls was done as per leaf

by manual counting. The gall formation rate was evaluated by an index, namely Air Pollution

Index (API) was postulated by following formula:

Air Pollution Index (API) = No. of galls per centimeter2 x Whole leaf L/B ratio

2. 4. Anatomical observation

The 10 leaves per tree were collected randomly. Individual leaf was cleaned properly in

running water and soaked with blotting paper. The anatomical observation was done under

bright field microscope (Olympus) with a 40x magnification.

2. 5. Statistical analysis

All the mean values of data were determined statistically significant differences

between experimental and control leaf samples for morphological features by using Student’s


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t-test (P < 0.05 level). The statistical analyses were done by using software (Microsoft Ver.

8.1, Excel 2013 with add on statistical tool pack).

3. RESULTS AND DISCUSSION

The present results clearly indicate that vehicular load brought significant changes in

foliar morphology, numbers of gall formation and anatomy of leaves and gall in A. scholaris

(Table 1 and Fig. 1, 2, 3, 4 and 6).

Fig 1a and b show significant changes in green colour indicating high chlorophyll

content in normal leaves in comparison to excessively affected bugs having yellowish green.

According to Albert et al. (2011), chlorophyll content is varied from new gall leaves to mature

gall leaves when compared to ungalled or less galled leaves.

In all experimental sites such as MVL, HVL and HeVL, the extra growth and

sometimes reduction pattern were significantly (P < 0.001, 0.01 or 0.05) observed when

compared to control site (LVL) for L, B and L/B ratio as morphological features (Table 1).

For the parameter L, significantly decreased values (P < 0.001) were observed for both area

viz. MVL and HVL but the value was also decreased in HeVL area at a significant level of P

< 0.05 when compared to LVL. In case of the parameter B, the values for both area viz. MVL

and HVL were decreased significantly (P < 0.01) but for HeVL decreased at a significant

level of P < 0.05 when compared to LVL. The values for L/B ratio were also decreased

significantly for all study area compared to control area. The important morphological

parameter as gall formation onto leaf lamina were showed an increasing trend at a significant

level of P<0.001. It was found the gall quantity with an increased value of 6 fold in MVL, 7

fold in HVL and 9 fold in HeVL in comparison to LVL (Table 1). Several researchers in

previous findings have documented the morphological deformities. According to them,

reduction in leaf area due to particulates pollution and particulates along with other air

pollutants such as O3, SO2 and NOx, PAN have more damaging effect on leaves (Joshi and

Swami 2007; Tiwari et al., 2008; Deepalakshmi, 2013; Nandy et al., 2014), which supports

the present study.

Table 1. Measurement of the shape of leaves due to the presence of galls

Sl. No. Area Parameters (n = 10; M ± SD)

L (cm) B (cm) L/B (cm) Gall quantity

(nos.)

1. LVL 23.8 ± 1.60 4.57 ± 0.53 5.20 ± 0.29 2.40 ± 2.17

2. MVL 12.69 ± 3.82* 3.51 ± 0.81** 3.55 ± 0.34* 12.30 ± 5.56*

3. HVL 15.84 ± 1.88* 3.94 ± 0.52** 4.02 ± 0.32* 14.3 ± 6.57*

4. HeVL 21.01 ± 4.74*** 3.97 ± 0.71*** 3.65 ± 0.49* 18.7 ± 7.04*

*P < 0.001; **P < 0.01; ***P < 0.05


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The visible injuries such as cholorosis, pigmentation, necrosis etc. in leaves were not

found in all the study area but pathogenesis may alter the colour of the leaves as green to

yellowish green (Fig. 1). All images in different types of external gall shapes viz. newly

formed, matured and perforated after releasing of bugs were depicted in Fig. 2 a, b and c.

Moreover, visible injuries are potent indication of air pollution and the parts and/or whole

plant showed susceptibility to individual and/or combination of air pollutants (Saquib et al.,

2010; Deepalakshmi, 2013; Nandy et al., 2014).

(a) (b)

Fig. 1. Morphological features along with galls (a) control leaf and (b) experimental leaf

(a)


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(b)

(c)

Fig. 2. Morphological features in experimental area: (a) newly formed galls, (b) matured galls

and (c) perforated galls after releasing bugs

The leaves of A. scholaris exist in whorls of seven in numbers. The leaf dorsal surface

is called as abaxial and the ventral surface is known as adaxial. In the present study, it was

observed that the leaves of experimental area were more yellowish green and normal green in

control area. The infected leaves were observed growth reduction with crumple in shape (Fig.

3a, b, c and d). It was also recorded that galls formation were found onto both surface of the

leaves while in some experimental area only from abaxial side, which is supported by Albert

et al. (2011). The study of whorls of A. scholaris is also an important parameter in

morphology.


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(a) Adaxial view and maximum numbers of newly formed galls in experimental leaves

(b) Abaxial view and maximum numbers of newly formed galls in experimental leaves


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(c) Adaxial view and less numbers of newly formed galls in control leaves

(d) Abaxial view and less numbers of newly formed galls in control leaves

Fig. 3. Morphological features of leaf whorl of experimental (a and b) and

control area (c and d)


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The tree, A. scholaris were found at a height of 40-45 feet (Fig. 5a). The present study

of gall formation on the leaves of Alstonia scholaris indicates that leaves below 10-15 feet

height were showed maximum numbers of galls while beyond 15 feet height no galls

observed near roadside of MVL, HVL and HeVL areas in comparison to LVL (Fig. 5a, b and

b). It is hypothesized that gaseous pollutants from vehicles may be diffused within 20foot

height. Several researchers have been documented that air pollutants cause changes in

morphology, anatomical features, growth rate, biochemical, physiological profiles and also

injuries on the leaves of trees (Iqbal et al., 1996; Ghosh et al., 1998; Viskari et al., 2000;

Oksanen and Holopainen, 2001; Peeters, 2002; Furlan et al., 2004; Nandy et al., 2014).

Fig. 4. Gall formation a particular height of tree.

It is also noted that API value was significantly higher (P < 0.001) as per gall

quantification in the experimental area compared to control area as represented by bar

diagram (Fig. 5). In our study, API values were found an increasing trend at significant level

in HVL and HeVL area but increased without significant level in MVL when compared to

LVL area. This is the first report that API can be an early detector of air pollutants to A.

scholaris. In addition, APTI (Air Pollution Tolerance Index) has already been documented by

other researchers, in which total chlorophyll, ascorbic acid, leaf extract pH and relative water

(a) A. scholaris tree (40-45 feet height)

(c) Newly formed galls within

10-15 feet height

(b) No galls formation beyond

15 feet height


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content were examined to know sensitivity of the trees towards air pollution (Singh and Rao

1983; Choudhury and Banerjee, 2009; Tripathi et al., 2009). In support, tree species, A.

scholaris has exhibited low APTI value and established a sensitive species to air pollutants

exposure. Altogether, our data revealed that increased numbers of gall formation and higher

API values in the leaves of A. scholaris may be suitable indicator of air pollution.

Fig. 5. Graphical representation of Air Pollution Index (API) versus vehicular load in

different study area (n = 10; M ± SD; *P < 0.001)

The anatomical features were found deformities both dorsal and ventral surface of

leaves along with the internal structure of gall. It was observed deformed epidermis and

palisade tissue when compared to control area and deformed gall shape (Fig 6a, b, c and d). In

the control leaf it was found cells are tubular, compact and covered with thick cuticle in upper

epidermis while papillae with thick cuticle in lower epidermis. It was also observed

hyperplasia in the palisade tissue. The present anatomical abnormality due to numerous gall

formations on experimental leaves is found closely similar work reported by Albert et al.

(2011). Moreover, it is suggested to visualize deformities under electron microscope, which

can be resulted in detailing cellular features.

(a) (b) (c) (d)

Fig. 6. Anatomical observations (in cross section): (a) control leaf, (b) leaf with early gall,

(c) deformed epidermis and palisade tissue in leaf having early formed gall and (d) deformed

shape of matured gall


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Air pollution can also induce qualitative and quantitative changes in secondary

metabolite composition (Lea et al., 1996; Zobel, 1996; Kanoun et al., 2001; Lopanen et al.,

2001). These secondary metabolites help as protection against insect herbivores, pests and

pathogens. According to Dicke, 2000; Gatehouse, 2002 and Furlan et al., 2004, abnormal

abiotic factors such as air pollutants have capability to change the structure and/or quantity of

secondary metabolites in leaves. In this context, air pollution can also inhibit carbon capture

capacity in plant and decrease nitrogen:carbon ratio, ultimately reduced the level of carbon-

based secondary compounds while enhanced the level of nitrogen-based compounds (Jones

and Coleman, 1991; Hamilton et al., 2001; Furlan et al., 2004). Another important

phenomenon e.g. fibers and lignin production require carbon that hamper due to the

alterations of secondary metabolites (Furlan et al., 2004). The present results are contradictory

that insect herbivory induced gall formation only by chemical stimuli in A. scholaris as

observed by Albert et al. (2011) and Mandal Biswas et al. (2014). However, there may be

alterations of secondary metabolites by air pollutants where insect herbivory affect the leaves

of tree, A. scholaris, subsequently abnormalities observed as reported by Gatehouse, (2002)

and Furlan et al., (2004).

It is further an interesting observation by Holopainen and Oksanen (1995) that arboreal

insects are suitable indicator of air pollution because these species found in maximum

numbers for host plant herbivory and parasitism can be easy due to increase level of nitrogen

content. It was documented that herbivorous insects with different feeding habits, respond to

stress-enhanced changes in their host trees with different intensity (Larsson, 1989). According

to Larsson, gall forming insects adapt to air pollution stress in host plant and are already

considered as air pollution indicator. It was established that gall-forming aphids such as

Sacchiphantes sp., Adelges sp., etc. have been reported with higher population densities in

zones of heavy air pollution (Ranft, 1968; Pfeffer, 1978).

4. CONCLUSION

In conclusion, this study is an assessment of host-plant herbivory and the gall

formation onto leaf, which is a can be a suitable indicator to know API index as a primary test

of air pollution. According to Central Pollution Control Board (CPCB), A. scholaris is a

suitable tree for greenbelt to mitigate air pollution but this tree species has documented as a

sensitive species among other plants in relation to air pollution (Singh and Rao 1983; Tripathi

et al., 2009). On the other hand, gall forming insect species is also found on the leaves of trees

during stress-induced condition, for example, air pollution (Holopainen and Oksanen, 1995).

Further researches should be needed to understand phytochemicals content alteration,

biochemical and genetic damages in A. scholaris along with abnormalities of gall forming

insects in vehicular loaded area.

Acknowledgement

The authors convey their thanks to Botany Hons. 2nd

year students Souvik and Souptik, Department of Botany,

Bangabasi and Sreerampore College, University of Calcutta, for help during sample collection for the present

study. Also thankful to Mr. Binayak Pal, Sr. Technical Officer, CSIR-Indian Institute of Chemical Biology,

Kolkata for photography of all images in present manuscript.


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( Received 05 July 2016; accepted 19 July 2016 )

Documents

Galls on Alstonia scholaris leaves as air pollution indicator … · Galls on Alstonia scholaris leaves as air pollution indicator Partha Talukdar1, Kaushiki Das2, Shrinjana Dhar2,